Inhibitors of farnesyl protein transferase

ABSTRACT

The present invention is directed to compounds which inhibit farnesyl-protein transferase (FTase) and the farnesylation of the oncogene protein Ras. The invention is further directed to chemotherapeutic compositions containing the compounds of this invention and methods for inhibiting farnesyl-protein transferase and the farnesylation of the oncogene protein Ras.

BACKGROUND OF THE INVENTION

The Ras gene is found activated in many human cancers, includingcolorectal carcinoma, exocrine pancreatic carcinoma, and myeloidleukemias. Biological and biochemical studies of Ras action indicatethat Ras functions like a G-regulatory protein, since Ras must belocalized in the plasma membrane and must bind with GTP in order totransform cells (Gibbs, J. et al., Microbiol. Rev. 53:171-286 (1989).Forms of ras in cancer cells have mutations that distinguish the proteinfrom Ras in normal cells.

At least 3 post-translational modifications are involved with Rasmembrane localization, and all 3 modifications occur at the C-terminusof Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or"Cys--Aaa¹ --Aaa² --Xaa" box (Aaa is an aliphatic amino acid, the Xaa isany amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Otherproteins having this motif include the Ras-related GTP-binding proteinssuch as Rho, fungal mating factors, the nuclear lamins, and the gammasubunit of transducin.

Farnesylation of Ras by the isoprenoid farnesyl pyrophosphate (FPP)occurs in vivo on Cys to form a thioether linkage (Hancock et al., Cell57:1167 (1989); Casey et al., Proc. Natl. Acad. Sci. USA 86:8323(1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formationof a thioester on a Cys residue near a C-terminal Cys farnesyl acceptor(Gutierrez et al., EMBO J. 8:1093-1098 (1989); Hancock et al., Cell 57:1167-1177 (1989)). Ki-Ras lacks the palmitate acceptor Cys. The last 3amino acids at the Ras C-terminal end are removed proteolytically, andmethyl esterification occurs at the new C-terminus (Hancock et al.,ibid). Fungal mating factor and mammalian nuclear lamins undergoidentical modification steps (Anderegg et al., J. Biol. Chem. 263:18236(1988); Farnsworth et al., J. Biol. Chem. 264:20422 (1989)).

Inhibition of Ras farnesylation in vivo has been demonstrated withlovastatin (Merck & Co., Rahway, N.J.) and compactin (Hancock et al.,ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). Thesedrugs inhibit HMG-CoA reductase, the rate limiting enzyme for theproduction of polyisoprenoids and the farnesyl pyrophosphate precursor.It has been shown that a farnesyl-protein transferase using farnesylpyrophosphate as a substrate is responsible for Ras farnesylation.(Reiss et al., Cell, 62: 81-88 (1990); Schaber et al., J. Biol. Chem.,265:14701-14704 (1990); Schafer et al., Science, 249: 1133-1139 (1990);Manne et al., Proc. Natl. Acad. Sci USA, 87: 7541-7545 (1990)).

Inhibition of farnesyl-protein transferase and, thereby, offarnesylation of the Ras protein, blocks the ability of Ras to transformnormal cells to cancer cells. The compounds of the invention inhibit Rasfarnesylation and, thereby, generate soluble Ras which, as indicatedinfra, can act as a dominant negative inhibitor of Ras function. Whilesoluble Ras in cancer cells can become a dominant negative inhibitor,soluble Ras in normal cells would not be an inhibitor.

A cytosol-localized (no Cys-Aaa¹ --Aaa² --Xaa box membrane domainpresent) and activated (impaired GTPase activity, staying bound to GTP)form of Ras acts as a dominant negative Ras inhibitor of membrane-boundRas function (Gibbs et al., Proc. Natl. Acad. Sci. USA86:6630-6634(1989)). Cytosol-localized forms of Ras with normal GTPaseactivity do not act as inhibitors. Gibbs et al., ibid, showed thiseffect in Xenopus oocytes and in mammalian cells.

Administration of compounds of the invention to block Ras farnesylationnot only decreases the amount of Ras in the membrane but also generatesa cytosolic pool of Ras. In tumor cells having activated Ras, thecytosolic pool acts as another antagonist of membrane-bound Rasfunction. In normal cells having normal Ras, the cytosolic pool of Rasdoes not act as an antagonist. In the absence of complete inhibition offarnesylation, other farnesylated proteins are able to continue withtheir functions.

Farnesyl-protein transferase activity may be reduced or completelyinhibited by adjusting the compound dose. Reduction of farnesyl-proteintransferase enzyme activity by adjusting the compound dose would beuseful for avoiding possible undesirable side effects such asinterference with other metabolic processes which utilize the enzyme.

These compounds and their analogs are inhibitors of farnesyl-proteintransferase. Farnesyl-protein transferase utilizes farnesylpyrophosphate to covalently modify the Cys thiol group of the Ras CAAXbox with a farnesyl group. Inhibition of farnesyl pyrophosphatebiosynthesis by inhibiting HMG-CoA reductase blocks Ras membranelocalization in vivo and inhibits Ras function. Inhibition offarnesyl-protein transferase is more specific and is attended by fewerside effects than is the case for a general inhibitor of isoprenebiosynthesis.

Previously, it has been demonstrated that tetrapeptides with the CAAXsequence inhibit Ras farnesylation (Schaber et al., ibid; Reiss et. al.,ibid; Reiss et al., PNAS, 88:732-736 (1991)). However, the reportedinhibitors of farnesyl-transferase are metabolically unstable orinactive in cells.

The compounds of the present invention, which contain one or morereduced peptide bonds, and are capable of forming a 5- or 6- memberedlactone or thiolactone ring, are inhibitors of Ras farnesyl-transferase.The presence of the reduced amide linkage confers metabolic stability tothese inhibitors such that they are capable of inhibiting Rasfarnesylation in vivo. Reduction of these amide bonds leads to anunexpected enhancement of intrinsic enzyme-inhibitory activity. Inaddition, the lactone forms of these inhibitors are prodrugs thatefficiently deliver the more active parent hydroxy or mercapto acids tothe intracellular compartment that is the site of Ras farnesylation.

It is, therefore, an object of this invention to develop compounds whichwill inhibit farnesyl-protein transferase and the farnesylation of theoncogene protein Ras. It is a further object of this invention todevelop chemotherapeutic compositions containing the compounds of thisinvention, and methods for producing the compounds of this invention.

SUMMARY OF THE INVENTION

The present invention includes compounds which inhibit farnesyl-proteintransferase and the farnesylation of the oncogene protein Ras,chemotherapeutic compositions containing the compounds of thisinvention, and methods for producing the compounds of this invention.

The compounds of this invention are illustrated by the formula: ##STR1##

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention are useful in the inhibition offarnesyl-protein transferase and the farnesylation of the oncogeneprotein Ras. In a first embodiment of this invention, the compounds areillustrated by the formula: ##STR2## wherein: X or Y are independentlyH₂ or O, provided that at least one of these is H₂ ;

R¹ is H₁ an alkyl group, an acyl group, an alkylsulfonyl group orarylsulfonyl group, wherein alkyl and acyl groups comprise straightchain or branched chain hydrocarbons of 1 to 6 carbons atoms, or in thealternative R¹ NH may be absent;

R² and R³ are the side chains of naturally occurring amino acids, or inthe alternative may be substitued or unsubstituted aliphatic, aromaticor heterocyclic groups, such as allyl, cyclohexyl, phenyl, pyridyl,imidazolyl or saturated chains of 2 to 8 carbon atoms, wherein thealiphatic substitutients may be substituted with an aromatic orheteroaromatic ring;

Z is O or S; and

n is 0, 1, or 2;

or the pharmaceutically acceptable salts thereof.

In a second embodiment of this invention, the compounds are illustratedby the formula: ##STR3## wherein: X or Y are independently H₂ or O,provided that at least one of these is H₂ ;

R¹ is H₁ an alkyl group, an acyl group, an alkylsulfonyl group orarylsulfonyl group, wherein alkyl and acyl groups comprise straightchain or branched chain hydrocarbons of 1 to 6 carbon atoms, or in thealternative R¹ NH may be absent;

R² and R³ are the side chains of naturally occurring amino acids, or inthe alternative may be substituted or unsubstituted aliphatic, aromaticor heterocyclic groups, such as allyl, cyclohexyl, phenyl, pyridyl,imidazolyl or saturated chains of 2 to 8 carbon atoms, wherein thealiphatic substituents may also be substituted with an aromatic orheteroaromatic ring;

Z is O or S; and

n is 0, 1, or 2;

or the pharmaceutically acceptable salts thereof.

The preferred compounds of this invention are as follows:

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanylhomoserine,

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucylhomoserine,

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanylhomoserine lactone,

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucylhomoserine lactone,

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanylhomocysteinelactone,

N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine lactone,

N-[N'-(2(R)-amino-3mercaptopropyl)isoleucyl-phenylalanyl]-3(S)-amino-tetrahydropyran-2-one,

N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-amino-tetrahydropyran-2-one,

N-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucylhomocysteine lactone,

N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine,

N-[N'-(2(R)-amino-3mercaptopropyl)isoleucyl-phenylalanyl]-3(S)-amino-4-hydroxypentanoicacid, or

N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucylisoleucyl]3(S)-amino-4-hydroxypentanoicacid.

The most preferred compounds of this invention are as follows:

N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine ##STR4##N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine lactone##STR5##N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-amino-4-hydroxy-pentanoicacid, or ##STR6##N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-aminotetrahydropyran-2-one.##STR7##

In the present invention, the amino acids are identified both byconventional 3 letter and single letter abbreviations as indicatedbelow:

    ______________________________________                                        Alanine            Ala         A                                              Arginine           Arg         R                                              Asparagine         Asn         N                                              Aspartic acid      Asp         D                                              Asparagine or      Asx         B                                              Aspartic acid                                                                 Cysteine           Cys         C                                              Glutamine          Gln         Q                                              Glutamic acid      Glu         E                                              Glutamine or       Glx         Z                                              Glutamic acid                                                                 Glycine            Gly         G                                              Histidine          His         H                                              Isoleucine         Ile         I                                              Leucine            Leu         L                                              Lysine             Lys         K                                              Methionine         Met         M                                              Phenylalanine      Phe         F                                              Proline            Pro         P                                              Serine             Ser         S                                              Threonine          Thr         T                                              Tryptophan         Trp         W                                              Tyrosine           Tyr         Y                                              Valine             Val         V                                              ______________________________________                                    

The pharmaceutically acceptable salts of the compounds of this inventioninclude the conventional non-toxic salts of the compounds of thisinvention as formed, e.g., from non-toxic inorganic or organic acids.For example, such conventional non-toxic salts include those derivedfrom inorganic acids such as hydrochloric, hydrobromic, sulfuric,sulfamic, phosphoric, nitric and the like: and the salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the compounds of this inventioncan be synthesized from the compounds of this invention which contain abasic moiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base with stoichiometric amounts or withan excess of the desired salt-forming inorganic or organic acid in asuitable solvent or various combinations of solvents.

The pharmaceutically acceptable salts of the acids of the compounds ofthis invention are also readily prepared by conventional procedures suchas treating an acid of the compounds of this invention with anappropriate amount of a base, such as an alkali or alkaline earth metalhydroxide e.g. sodium, potassium, lithium, calcium, or magnesium, or anorganic base such as an amine, e.g., dibenzylethylenediamine,trimethylamine, piperidine, pyrrolidine, benzylamine and the like, or aquaternary ammonium hydroxide such as tetramethylammonium hydroxide andthe like.

The compounds of the invention can be synthesized from their constituentamino acids by conventional peptide synthesis techniques, and additionalmethod described below. Standard methods of peptide synthesis aredisclosed, for example, in the following works: Schroeder et al., "ThePeptides", Vol. I, Academic Press 1965, or Bodanszky et al., "PeptideSynthesis", Interscience Publishers, 1966, or McOmie (ed.) "ProtectiveGroups in Organic Chemistry", Plenum Press, 1973, or Barany et al., "ThePeptides: Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press,1980, or Stewart et al., "Solid Phase Peptide Synthesis", SecondEdition, Pierce Chemical Company, 1984. The teachings of these works arehereby incorporated by reference.

The compounds of this invention are prepared according to the reactionSchemes as set forth below: ##STR8##

Compounds of this invention are prepared by employing reactions A-C asshown in Schemes 1-3 above, in addition to other standard manipulationssuch as ester hydrolysis, cleavage of peptide protecting groups, etc.,as may be known in the literature or exemplified in the Examples. Thekey bond-forming reactions are as follows:

Reaction A. Peptide bond formation and protecting group cleavage usingstandard solution or solid phase methodologies.

Reaction B. Preparation of a reduced subunit by borane reduction of theamide moiety.

Reaction C. Preparation of a reduced peptide subunit by reductivealkylation of an amine by an aldehyde using sodium cyanoborohydride,hydrogen and a catalyst or other reducing agents.

These reactions may be employed in a linear sequence to provide thecompounds of the invention or they may be used to synthesize dipeptidefragments which are subsequently joined by the alkylation or acylationreactions described in the Schemes.

The compounds of this invention inhibit farnesyl-protein transferase andthe farnesylation of the oncogene protein Ras. These compounds areuseful as pharmaceutical agents for mammals, especially for humans.These compounds may be administered to patients for use in the treatmentof cancer. Examples of the type of cancer which may be treated with thecompounds of this invention include, but are not limited to, colorectalcarcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.

The compounds of this invention may be administered to mammals,preferably humans, either alone or, preferably, in combination withpharmaceutically-acceptable carriers or diluents, optionally with knownadjuvants, such as alum, in a pharmaceutical composition, according tostandard pharmaceutical practice. The compounds can be administeredorally or parenterally, including intravenous, intramuscular,intraperitoneal, subcutaneous, rectal and topical administration.

For oral use of a chemotherapeutic compound according to this invention,the selected compounds may be administered, for example, in the form oftablets or capsules, or as an aqueous solution or suspension. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch, and lubricating agents, such as magnesiumstearate, are commonly added. For oral administration in capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents may be added. For intramuscular,intraperitoneal, subcutaneous and intravenous use, sterile solutions ofthe active ingredient are usually prepared, and the pH of the solutionsshould be suitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled in order to render thepreparation isotonic.

The present invention also encompasses a pharmaceutical compositionuseful in the treatment of cancer, comprising the administration of atherapeutically effective amount of the compounds of this invention,with or without pharmaceutically acceptable carriers or diluents.Suitable compositions of this invention include aqueous solutionscomprising compounds of this invention and pharmacologically acceptablecarriers, e.g. saline, at a pH level, e.g., 7.4. The solutions may beintroduced into a patient's intramuscular blood-stream by local bolusinjection.

When a compound according to this invention is administered into a humansubject, the daily dosage will normally be determined by the prescribingphysician with the dosage generally varying according to the age,weight, and response of the individual patient, as well as the severityof the patient's symptoms.

In one exemplary application, a suitable amount of compound isadministered to a human patient undergoing treatment for cancer.Administration occurs in an amount between about 0.1 mg/kg of bodyweight to about 20 mg/kg of body weight of a mammal per day, preferablyof between 0.5 mg/kg of body weight to about 10 mg/kg of body weight ofa mammal per day.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limitativeof the reasonable scope thereof.

EXAMPLE 1 Preparation ofN-(2(R)-Amino-3-mercaptopropyl)isoleucyl-phenylalanyl-homoserine lactoneand N-(2(R)-Amino-3-mercaptopropyl)isoleucyl-phenylalanyl-homoserineStep A: Preparation of (t-Butoxycarbonyl)phenylalanyl-homoserine lactone

To a solution of N-t-butoxycarbonylphenylalanine (1.69 g, 6.39 mmol) inCH₂ Cl₂ (10 mL) and EtOAc (10 mL) were added3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT, 1.04 g, 6.39mmol) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC, 1.23 g,6.39 mmol) followed by the hydrochloride salt homoserine lactone (0.80g, 5.81 mmol). The pH was adjusted to 6.5-7.0 withN,N-diisopropylethylamine (1.11 mL, 6.39 mmol) and the mixture wasstirred at ambient temperature for 16 hours. The mixture wasconcentrated and the residue was partitioned between EtOAc (100 mL) andH₂ O (50 mL). The organic layer was washed with 10% citric acid (1×25mL), saturated NaHCO₃ (1×25 mL), brine (1×25 mL), dried (Na₂ SO₄),filtered, and concentrated. The crude product was purified bychromatography (silica gel, CH₂ Cl₂ : MeOH, 98:2) to give 1.4 g of thetitle compound.

Step B: Phenylalanyl-homoserine lactone hydrochloride salt

The product of Step A (1.4 g, 4.02 mmol) was dissolved in EtOAc (40 mL),cooled to -25° C., and treated with gaseous HCl (10 min.) followed bynitrogen (10 min.) to remove excess HCl. The solvent was evaporated togive 1.14 g of the title compound.

Step C: Preparation of N-(t-butoxycarbonyl)-S-triphenylmethylcysteinealdehyde

This compound was synthesized by applying the procedure of Goel, Krolls,Stier, and Kesten to N-(t-butoxycarbonyl)-S-trityl cysteine (Org.Synthesis 67, 69 (1988). The compound was obtained as a white solid,which was used without purification. ¹ H NMR (CDCl₃) δ 9.2 (1H, s),7.5-7.1 (18H, m), 5.1 (1H, br d), 3.92 (1H, m), 2.85-2.5 (2H, m), 1.4(9H, s).

Step D: N-[(2R)-(t-Butoxycarbonylamino)-3-triphenylmethylmercaptopropyl]isoleucine

Isoleucine (1.97 g, 0.015 mol) was suspended in EtOH (150 mL) withN-t-butoxycarbonyl-S-triphenylmethylcysteine aldehyde (6.71 g, 0.015mol) and 3A molecular sieves. Sodium cyanoborohydride (0.47 g, 0.0075mol) was added and the mixture was stirred at ambient temperature for 72hours. Filtration and concentration gave an oil, which waschromatographed (silica gel, CH₂ Cl₂ : MeOH, 95:5 to 9:1) to give 2.1 gof the title compound, mp 83°-90° C. ¹ H NMR (CDCl₃) δ 7.19-7.41 (m,15H), 4.98-5.12 (m, 1H), 3.58-3.70 (m, 2H), 3.18 (br s, 1H), 2.78-2.81(m, 2H), 2.32-2.60 (m, 2H), 1.80-1.96 (m, 1H), 1.40 (s, 9H), 1.20-1.35(m, 1H), 0.84-0.93 (m, 6H).

Step E: N-[2(R)-(t-Butoxycarbonyl)amino-3-(triphenylmethyl)mercaptopropyl]isoleucyl-phenylalanyl-homoserine lactone

N-[2(R)-(t-butoxycarbonyl)amino-3-(triphenylmethyl)mercaptopropyl]isoleucine (0.30 g, 0.53 mmol), dissolved in CH₂ Cl₂ (10mL) and EtOAc (10 mL), was treated with HOOBT (96 mg, 0.59 mmol), EDC(0.112 g, 0.59 mmol), and phenylalanyl-homoserine lactone hydrochloridesalt (0.167 g, 0.59 mmol). The pH was adjusted to 6.5-7.0 withN,N-diisopropylethylamine (0.102 mL, 0.59 mmol) and the mixture wasstirred at ambient temperature for 16 hours. The mixture wasconcentrated and the residue was partitioned between EtOAc (30 mL) andH₂ O (15 mL). The organic layer was washed with 10% citric acid (1×15mL), saturated NaHCO₃ (1×15 mL), brine (1×15 mL), and dried (Na₂ SO₄).Filtration and concentration gave the crude product, which waschromatographed twice (silica gel, CH₂ Cl₂ : MeOH, 98:2; silica gel,EtOAc: hexane, 2:1) to provide 0.115 g of the title compound.

Step F: N-(2(R)-Amino-3-mercaptopropyl) isoleucylphenylalanyl-homoserinelactone

The product of Step E was dissolved in CH₂ Cl₂ (2 mL) andtrifluoroacetic acid (1 mL) was added followed by triethylsilane (0.093mL, 0.58 mmol). The mixture was stirred at ambient temperature for 1 h,concentrated, and the residue was triturated with Et₂ O to give 0.078 gof pure title compound, m.p. 103°-105° C. ¹ H NMR (DMSO) δ 8.75 (d,J=9Hz, 1H), 7.34-7.17 (m, 5H), 4.75-4.53 (m, 2H), 4.43 (t, J=18 Hz, 1H),4.28-4.16 (m, 1H), 3.30-3.16 (m, 1H), 3.04 (dd,J=12,14 Hz, 1H), 2.85(dd,J=12,14 Hz, 1H), 2.75-2.33 (m, 7H), 2.20-2.05 (m, 1H), 1.66-1.42 (m,2H), 1.16-1.00 (m, 1H), 0.89-0.70 (m, 6H). Anal. Calcd for C₂₂ H₃₄ N₄ O₄S. 2CF₃ CO₂ H: C, 46.02; H, 5.35; N, 8.26. Found: C, 46.19; H, 5.23; N,8.41.

Step G: N-(2(R)-Amino-3-mercaptopropyl) isoleucylphenylalanyl-homoserine

N-(2(R)-Amino-3-mercaptopropyl) isoleucyl-phenylalanyl-homoserinelactone (0.003 g, 0.004 mmol) was dissolved in MeOH (0.1 mL) and 1N NaOH(0.013 mL) was added followed by MeOH (0.305 mL). Conversion of thelactone to the hydroxy-acid was confirmed by HPLC analysis and ¹ H NMRspectroscopy.

EXAMPLE 2 Preparation ofN-(2(R)-Amino-3-mercaptopropyl)isoleucyl-isoleucyl-homersine lactone andN-(2(R)-Amino-3-mercaptopropyl)-isoleucyl-isoleucyl-homoserine

The title compounds were prepared according to the methods of Example 1,substituting N-t-butoxycarbonyl-isoleucine for the phenylalaninederivative used in Step A. The lactone was obtained as a solid, mp111°-113° C. ¹ H NMR (DMSO) δ 8.66 (d,J=9 Hz, 1H), 8.49-8.28 (m, 1H),4.61 (q, J=9 Hz, 1H), 4.36 (t, J=9 Hz, 1H), 4.31-4.15 (m, 2H), 3.50-3.34(m, 2H), 3.00-2.71 (m, 4H), 2.45-2.30 (m, 1H), 2.30-2.17 (m, 1H),1.85-1.4 (m, 5H), 1.22-1.05 (m, 2H), 0.97-0.74 (m, 12H). Anal. Calcd forC₁₉ H₃₆ N₄ O₄ S . 2 CF₃ COOH: C, 42.85; H, 5.94; N, 8.69. Found: C,43.00; H, 5.69; N, 8.89. The hydroxy acid was generated in situaccording to Example 1, Step G.

EXAMPLE 3 Preparation ofN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-aminotetrahydropyran-2-oneandN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-amino-4-hydroxy-pentanoicacid Step A: Preparation of 3(S)-aminotetrahydropyran-2-one.

The method of Gong and Lynn (J. Org. Chem. 55, 4763 (1990)) was used toconvert L-glutamic acid to 3(S)-amino-4-hydroxy-pentanoic acid. Thecrude product of this reaction was treated with di-t-butyl dicarbonateto obtain 3(S)-t-butoxycarbonylamino-4-hydroxy-pentanoic acid, which wasconverted to the title compound by reaction with EDC. The compound waspurified by column chromatography on silica gel. ¹ H NMR (CDCl₃) δ 5.35(1H, br s), 4.40 (m, 1H), 4.35 (2H, t, J=6 Hz), 2.60 (1H, m), 1.61 (1H,m), 1.47 (9H, s).

Step B: Preparation ofN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-aminotetrahydropyran-2-one

The product of Step A was converted to 3(S)-aminotetrahydropyran-2-onehydrochloride by treatment with HCl gas according to the method ofExample 1, Step B. This intermediate was further transformed to thetitle compound using the methods of Example 1: mp 88-93. Anal. Calcd forC₂₀ H₃₈ N₄ O₄ S. 2 CF₃ COOH. 0.5 H₂ O: C, 43.17; H, 6.19; N, 8.39.Found: C, 43.19; H, 6.34; N, 8.59.

The lactone was converted to the hydroxy acid by the method of Example1, Step G.

EXAMPLE 4 Preparation ofN-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserinelactone Step A: Preparation ofN-[2(S)-t-butoxycarbonylamino-3(S)-methylpentyl]isoleucyl homoserinelactone.

Isoleucyl homoserine was reductively alkylated withN-t-butoxycarbonyl-isoleucine aldehyde using the method of Example 1,Step D.

Step B: Preparation ofN-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserinelactone andN-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine.

The product of Step A was converted to the title lactone using themethods of Example 1, Steps D-F. The compound was obtained as a solid,mp 65°-69° C. Anal. Calcd for C₁₉ H₃₈ N₄ O₃ S . 3 CF₃ COOH: C, 39.60; H,5.65; N, 7.39. Found: C, 39.55; H, 5.45; N, 7.52.

The hydroxy acid was prepared in situ according to Example 1, Step G.

EXAMPLE 5 Preparation ofN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanyl]-3(S)-amino-tetrahydropyran-2-oneandN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucylphenylalanyl]-3(S)-amino-4-hydroxypentanoicacid

The lactone form of the title compound was prepared using the procedurein Example 3, employing phenylalanine in place of isoleucine in theappropriate Step. The compound was isolated as a solid, mp 95°-100° C.Anal. Calcd for C₂₂ H₃₆ N₄ O₄ S. 2 CF₃ COOH. 0.25 Et₂ O: C, 47.28; H,5.74; N, 7.88. Found: C, 47.63; H, 5.85; N, 8.11.

The hydroxy acid was prepared in situ according to Example 1, Step G.

EXAMPLE 6 Preparation ofN-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanyl-homocysteinelactone Step A: Preparation of isoleucyl-phenylalanyl-homocysteinelactone

N-t-butoxycarbonylisoleucyl-phenylalanine (496 mg) and3-hydroxy-4-oxo-1,2,3-benzotriazine (320 mg) were dissolved in a mixtureof DMF and methylene chloride and EDC (275 mg) was added. After 5 minhomocysteine thiolactone hydrochloride (204 mg) and N-methyl morpholine(310 μl) were added. The reaction was stirred for 16 hours at roomtemperature and the solvent was removed in vacuo. The residue waspartitioned between ethyl acetate and 10% citric acid solution. Theorganic phase was washed with saturated sodium bicarbonate solution andbrine, dried (Na₂ SO₄), filtered, and concentrated in vacuo. The solidresidue was chromatographed on silica gel to give a white solid product.This solid was dissolved in cold 25% trifluoroacetic acid: methylenechloride. After 45 min the reaction mixture was concentrated in vacuoand the residue was purified by preparative reverse phase HPLC.Lyophilization gave the title compound as a white solid. NMR (CDCl₃ +CD₃OD) δ 0.86 (m, 6H), 1.14 (m, 1H), 1.45 (m, 1H), 1.60-2.12 (br m, 7H),2.18 (m, 1H), 2.48 (m, 1H), 3.05 (m, 1H), 3.12-3.34 (m, 3H), 3.86 (d,1H), 4.35 (dd, 1H), 4.60 (m, 1H), 7.24 (m, 5H), 7.71 (d, 1H), 8.20 (d,1H).

Step B: Preparation ofN-[2(R)-amino-3-mercaptopropyl]isoleucyl-phenylalanyl homocysteinelactone

N-(t-butoxycarbonyl)-S-triphenylmethylcysteine aldehyde (188 mg) asprepared in Example 1, Step C, and the product of Step A (201.8 mg) weredissolved in anhydrous ethanol (5 ml) under an argon atmosphere. 3 Åmolecular sieves and 210 μl of 1M sodium cyanoborohydride in THF wereadded. The reaction mixture was stirred 16 hours, filtered andconcentrated in vacuo. The residue was chromatographed on silica gel togive N-[2(R)-(t-butoxycarbonylamino)-3-triphenylmethylmercaptopropyl]isoleucyl-phenylalanyl-homocysteinelactone as a solid intermediate. Further transformation by the methoddescribed in Example 1, Step F gave the title compound as a white solid,mp 82°-108° C. NMR (CD₃ OD) δ 0.76 (d, 3H), 0.86 (t, 3H), 1.09 (m, 1H),1.48 (m, 1H), 1.58 (m, 1H), 2.20 (m, 1H), 2.58 (m, 2H), 2.68 (m, 2H),2.78 (dd, 1H), 2.94 (m, 2H), 3.22 (m, 2H), 3.45 (m, 1H), 4.63 (dd, 1H),7.24 (m, 1H), 7.30 (m, 4H). Anal. Calcd for C₂₂ H₃₄ N₄ O₃ S₂.2CF₃ CO₂ H:C, 44.95; H, 5.22; N, 8.06. Found: C, 44.54; H, 4.97; N, 8.13.

EXAMPLE 7 Preparation ofN-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl-homocysteine lactone

Using the methods described in Example 2, substituting homocysteine forhomoserine, the title compound was obtained as a lyophilized powder, mp110°-112.7° C. ¹ H NMR (CD₃ OD) δ 0.94 (m, 9H), 1.02 (d, 3H), 1.23 (m,2H), 1.62 (m, 2H), 1.73 (m, 1H), 1.87 (m, 1H), 2.22 (m, 1H), 2.54 (m,1H), 2.78 (dd, 1H), 2.86 (m, 3H), 3.08 (d, 1H), 3.41 (m, 1H), 4.30 (m,1H), 4.62 (dd, 1H). Anal. Calcd for C₁₉ H₃₆ N₄ O₃ S₂.2CF₃ CO₂ H.0.8H₂ O:C, 40.92; H, 5.91; N, 8.30. Found: C, 40.86; H, 5.75; N, 8.49.

EXAMPLE 8 In vivo ras farnesylation assay

The cell line used in this assay was the v-ras line, which expressedviral Ha-ras p21. The assay was performed essentially as described inDeClue, J. E. et. al., Cancer Reasearch 51, 712-717, (1991). Cells in 10cm dishes at 50-75% confluency were treated with the test compound(final concentration of solvent, methanol or dimethyl sulfoxide, was0.1%). After 4 hours at 37° C., the cells were labelled in 3 mlmethionine-free DMEM supplemented with 10% regular DMEM, 2% fetal bovineserum and 400 μCi[³⁵ S] methionine (1000 Ci/mmol). After an additional20 hours, the cells were lysed in 1 ml lysis buffer (1% NP40/20 mMHEPES, pH 7.5/5 mM MgCl₂ /1 mM DTT/10 μg/ml aprotinen/2 μg/mlleupeptin/2 μg/ml antipain/0.5 mM PMSF) and the lysates cleared bycentrifugation at 100,000× g for 45 min. Aliquots of lysates containingequal numbers of acidprecipitable counts were brought to 1 ml with IPbuffer (lysis buffer lacking DTT) and immunoprecipitated with theras-specific monoclonal antibody Y13-259 (Furth, M. E. et al., J. Virol.43, 294-304 , (1982)). Following a 2 hour antibody incubation at 4° C.,200 μl of a 25% suspension of protein A-Sepharose coated with rabbitanti rat IgG was added for 45 min. The immunoprecipitates were washedfour times with IP wash buffer (20 mM HEPES, pH 7.5/1 mM EDTA/ 1% TritonX-100/0.5% deoxycholate/0.1% SDS/0.1M NaCl), boiled in SDS-PAGE samplebuffer and loaded on 13% acrylamide gels. When the dye front reached thebottom, the gel was fixed, soaked in Enlightening, dried andautoradiographed. The intensities of the bands corresponding tofarnesylated and nonfarnesylated ras proteins were compared to determinethe percent inhibition of farnesyl transfer to protein.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Ras farnesylation by compounds of this                          invention in the v-ras cell line                                              Compound                Inhibition                                            ______________________________________                                        N-[2(S)-(2(R)-amino-3-mercaptopropyl-                                                                 No Inhibition                                         amino)-3(S)-methylpentyl]isoleucyl-                                           homoserine                                                                    N-[2(S)-(2(R)-amino-3-mercaptopropyl-                                                                 90% Inhibition                                        amino)-3(S)-methylpentyl]isoleucyl-                                                                   at 100 μM test                                     homoserine lactone      concentration                                         ______________________________________                                    

EXAMPLE 9 In vitro inhibition of Ras Farnesyl Transferase

Farnesyl-protein transferase (FTase) from bovine brain waschromatographed on DEAE-Sephacel (Pharmacia, 0-0.8M NaCl gradientelution), N-octyl agarose (Sigma, 0-0.6M NaCl gradient elution), and amono Q HPLC column (Pharmacia, 0-0.3M NaCl gradient). Ras-CVLS at 3.5μM, 0.25 μM [³ H]FPP, and the indicated compounds were incubated withthis partially purified enzyme preparation. The FTase data presentedbelow is a measurement of the ability of the test compound to inhibitRas farnesylation in vitro.

                  TABLE 2                                                         ______________________________________                                        Inhibition of Ras farnesylation by compounds of this                          invention                                                                     Compound               IC.sub.50 *(nM)                                        ______________________________________                                        N-[2(S)-(2(R)-amino-3-mercaptopropyl-                                                                 12                                                    amino)-3(S)-methylpentyl]isoleucyl-                                           homoserine                                                                    N-[2(S)-(2(R)-amino-3-mercaptopropyl-                                                                950                                                    amino)-3(S)-methylpentyl]isoleucyl-                                           homoserine lactone                                                            N-[N'-(2(R)-amino-3-mercaptopropyl)-                                                                  24                                                    isoleucyl-isoleucyl]-3(S)-amino-4-                                            hydroxy-pentanoic acid                                                        N-[N'-(2(R)-amino-3-mercaptopropyl)-                                                                 470                                                    isoleucyl-isoleucyl]-3(S)-aminotetra-                                         hydropyran-2-one                                                              ______________________________________                                         *(IC.sub.50 is the concentration of compound which gives 50% inhibition o     FTase under the described assay conditions)                              

What is claimed is:
 1. A compound which inhibits farnesyl-proteintransferase of the formula: ##STR9## wherein: X or Y are independentlyH₂ or O, provided that at least one of these is H₂ ;R¹ is H, an alkylgroup, an acyl group, an alkylsulfonyl group or arylsulfonyl group,wherein alkyl and acyl groups comprise straight chain or branched chainhydrocarbons of 1 to 6 carbon atoms, or in the alternative R¹ NH may beabsent; R² and R³ are the side chains of naturally occurring aminoacids, or in the alternative may be substituted or unsubstitutedaliphatic, aromatic or heterocyclic groups, which comprise allyl,cyclohexyl, phenyl, pyridyl, imidazolyl or saturated chains of 2 to 8carbon atoms, wherein the aliphatic substitutents may be substitutedwith an aromatic or heteroaromatic ring; Z is O or S; and n is 0, 1, or2;or the pharmaceutically acceptable salts thereof.
 2. A compound whichinhibits farnesyl-protein transferase whichis:N-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenylalanylhomoserine,N-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl-homoserine,N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine,N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-phenyl-alanyl]-3(S)-amino-4-hydroxypentanoicacid, orN-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-amino-4-hydroxypentanoicacid, or the pharmaceutically acceptable salts thereof.
 3. A compound ofclaim 2 which is:N-[2(S)-(2(R)-amino-3-mercaptopropylamino)-3(S)-methylpentyl]isoleucyl-homoserine ##STR10##
 4. A compound of claim 2is:N-[N'-(2(R)-amino-3-mercaptopropyl)isoleucyl-isoleucyl]-3(S)-amino-4-hydroxy-pentanoicacid ##STR11##
 5. A pharmaceutical composition comprising apharmaceutical carrier, and dispersed therein, a therapeuticallyeffective amount of a compound of claim
 1. 6. A pharmaceuticalcomposition comprising a pharmaceutical carrier, and dispersed therein,a therapeutically effective amount of compound of claim
 2. 7. Apharmaceutical composition comprising a pharmaceutical carrier, anddispersed therein, a therapeutically effective amount of compound ofclaim
 3. 8. A pharmaceutical composition comprising a pharmaceuticalcarrier, and dispersed therein, a therapeutically effective amount ofcompound of claim 4.